This application is a renewal of the original grant that supports gap junction research in our laboratory. The overriding hypothesis of the proposed research is that cardiac myocytes regulate intercellular coupling by rapidly changing the number of functional channels within gap junctions primarily through posttranslational mechanisms involving changes in channel protein trafficking, assembly and degradation mediated by changes in connexin phosphorylation. Accordingly, the goal of the proposed research is to define the responsible chemical mediators, signaling pathways and phosphorylation events. In experiments designed to fulfill Specific Aim 1, we will define the contributions of major chemical mediators of stretch-induced up-regulation of Cx43 expression in cultured cardiac myocytes and elucidate the roles of changes in connexin synthesis, degradation and intracellular trafficking in this process. Studies in Aim 2 will focus on early responses to mechanical stretch which, we propose, are mediated by vascular endothelial growth factor (VEGF). We will identify signal transduction pathways activated by VEGF and determine how they control Cx43 trafficking and degradation through specific changes in Cx43 phosphorylation. In Specific Aim 3, we will investigate the role of myocyte-extracellular matrix interactions and integrin signaling in stretch-induced up-regulation of Cx43. We will define the relationship between mechanotransduction via integrin activation of focal adhesion kinase (FAK) and up-regulation of Cx43 expression. We will also define the relationship between FAK and VEGF signaling in the cascade of events ultimately leading to enhanced intercellular coupling. Studies in Specific Aim 4 are designed to elucidate mechanisms responsible for rapid uncoupling during acute ischemia. We will define the effects of ischemia and ischemic preconditioning on Cx43 expression, phosphorylation and channel function, and test the hypothesis that activation of calcineurin pathways and/or phosphorylation of Cx43 by protein kinase C mediates electrical uncoupling in response to acute ischemia. Insights gained through these studies will advance our knowledge of how the heart regulates intercellular communication and may identify new therapeutic targets to modulate coupling and, thereby, limit arrhythmogenesis or myocardial injury.